Kanazawa University NanoLSI Podcast

D. Carlero et al, ACS Nano 2024, 18, 30, 19518–19527

Researchers from Kanazawa University's NanoLSI, IMDEA Nanoscience, and CNB-CSIC studied influenza A replication using high-speed atomic force microscopy. They observed that recombinant ribonucleoprotein complexes (rRNPs) can undergo multiple transcription cycles, with RNA structure stability influencing synthesis rates. Their findings offer new insights into viral replication mechanisms and RNA synthesis regulation, opening doors for further research on gene expression control. 

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M.S. Alam et al, Small Methods 2024, 2400287

Atomic force microscopy (AFM) was initially developed to visualize surfaces at nanoscale resolution. Researchers at WPI NanoLSI, Kanazawa University, have now extended AFM for 3D imaging, particularly for flexible nanostructures like carbon nanotubes. They demonstrated that dynamic mode AFM, which uses a vibrating tip, causes less friction and damage than static mode, making it ideal for imaging delicate biological systems like cells, organelles, and vesicles​. 

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Madhu Biyani and colleagues at NanoLSI, Kanazawa University, have developed a sensitive and selective electrochemical biosensor for detecting the cancer biomarker ADAR1, using new aptamers. This cost-effective tool enables rapid ADAR1 detection in diluted samples, promising improved cancer prognosis and monitoring.

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Yanjun Zhang, Yuri Korchev, and colleagues used hopping probe scanning ion conductance microscopy to study hydrogen peroxide eustress on colorectal cancer cells, revealing varying cell stiffness and gradients. Their findings could lead to new cancer and inflammatory disease therapies. 

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Mikihiro Shibata and collaborators used high-speed atomic force microscopy to study nucleosome dynamics. They found that nucleosomes without histone tails, particularly H2B and H3, showed increased sliding and DNA unwrapping. These findings highlight the importance of histone tails in chromatin stability and structure. 

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Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Japan, collaborating with Professor Sarikaya, Seattle, USA.

The research described in this podcast was published in Small in February 2024

Kanazawa University NanoLSI website

https://nanolsi.kanazawa-u.ac.jp/en/

NanoLSI Podcast website

Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Holger Flechsig and Clemens Franz from WPI-NanoLSI, at Kanazawa University, in collaboration with Vincent Torre from the International School of Advanced Studies in Italy and former WPI-NanoLSI members Leonardo Puppulin and Arin Marchesi.

The research described in this podcast was published in Nature Communications in January 2024

Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/

Researchers observe the structural heterogeneity of a lipid scramblase

Researchers from Nano Life Science Institute (WPI-NanoLSI), at Kanazawa University report in Nature Communications that TMEM16F, a transmembrane protein that facilitates the passive movement of phospholipids and ions across membranes, explores a larger conformational landscape than previously thought to perform its unique functions. The finding refines our molecular understanding of crucial physiological processes such as blood coagulation and COVID-19 pathogenesis, and highlights the importance of probing membrane proteins in native-like environments.

Lipid scramblases are proteins embedded in cell membranes that play a crucial role in shuffling phospholipids between the two lipid layers that form such cellular boundaries. TMEM16F, a member of the TMEM16 protein family, acts as both a calcium-activated ion channel and a lipid scramblase, meaning that it can facilitate the transfer of both, lipids and ions across the chemical environment outside and inside of the cell. These movements regulate several biological functions such as blood clotting, bone development, and viral entry and are therefore of great physiological and clinical interest. At the molecular level, the TMEM16F architecture has a double-barrelled shape in which two identical polypeptide chains (called subunits), each formed by ten transmembrane (TM) helices, stick together (a process known as dimerization) to form two separate and presumably independent ion and lipid pathways.

Previously, it was thought that TMEM16F might work like a simple gate, with calcium ions serving as keys to unlock the two permeation pathways. Opening and closing the gate to different extents would let lipids and ions cross the plasma membrane alternately. However, structural investigations using cryo-electron microscopy (cryo-EM) -an in vitro technique that can reveal the 3D architecture of purified and frozen proteins at near-atomic resolution – have mostly captured TMEM16F snapshots in inactive conformations, with the ion and lipid gates presumably trapped in a closed state, raising questions about the validity of existing models.

So how did the researchers set about shedding light on how TMEM16F works?

To gain a better understanding of TMEM16F’s structure and function relationship, Holger Flechsig and Clemens Franz from WPI-NanoLSI, Kanazawa University, in collaboration with Vincent Torre from the International School of Advanced Studies (Italy) and former WPI-NanoLSI members Leonardo Puppulin and Arin Marchesi, used advanced techniques such as single-molecule force spectroscopy (SMFS) and high-speed atomic force microscopy (HS-AFM) imaging. These methods allowed them to observe TMEM16F behaviour at the molecular level in physiological environments, providing insights into its structure, dynamics, and mechanical properties.

The study uncovered that TMEM16F exhibits a wide range of structural conformations that have been overlooked so far. The research revealed unexpected changes in the dimerization interface and TMEM16F subunit arrangements, suggesting that TMEM16F operates in a more dynamic and versatile manner than previously thought. The authors propose that these large structural variations are critical for TMEM16F’s diverse functions, including lipid scrambling an

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Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Tareg Omer Mohammed, You-Rong Lin, and Clemens M. Franz at the Nano Life Science Institute (WPI-NanoLSI), at Kanazawa University.

The research described in this podcast was published in the Journal of Cell Science in January 2024.

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https://nanolsi.kanazawa-u.ac.jp/en/

A novel role for S100A11 in focal adhesion regulation

Researchers at Kanazawa University report in the Journal of Cell Science on a novel role of the small Ca2+ion-binding protein S100A11 [S one hundred A eleven] in focal adhesion disassembly.

S100A11 is a small Ca2+ion-activatable protein with an established role in different cellular processes involving actin cytoskeleton remodeling, such as cell migration, membrane protrusion formation, and plasma membrane repair. It also displays F-actin binding activity and localizes to actin stress fibers, but its precise role in regulating these structures remained unclear.

In their study, Tareg Omer Mohammed, You-Rong Lin, and Clemens M. Franz together with colleagues from the Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, in Japan, and Karlsruhe Institute of Technology, in Germany, report a novel localization of S100A11 to disassembling focal adhesions at the end of contractile stress fibers in HeLa and U2OS cells. Specifically, S100A11 transiently appears at the onset of focal adhesion disassembly, reliably marking the targeted adhesion sites for subsequent disassembly. Interestingly, S100A11 leaves focal adhesion sites before the completion of disassembly, indicating that S100A11 plays a specific role in the initiation of adhesion site disassembly, rather than the disassembly process itself.

So what are focal adhesions anyway and what can we learn from them?

Focal adhesions are integrin-containing cell/matrix adhesion sites enabling cells to adhere to the cellular environment and to apply cellular contraction forces during extracellular matrix remodeling. Directed cell migration requires the coordinated assembly of new adhesion sites at the front, and disassembly at the rear of the cell, and better understanding mechanisms regulating focal adhesion turnover is, therefore, an important goal in cell migration and invasion research. The newly discovered role of S100A11 in focal adhesion disassembly extends insights into the molecular mechanisms underlying focal adhesion site disassembly.

The authors furthermore delineate a force-dependent recruitment mechanism for S100A11 to adhesion sites involving non-muscle myosin II-driven stress fiber contraction, activation of mechanosensitive, Ca2+ ion-permeable Piezo1 channels, and intracellular Ca2+ ion influx at mechanically stressed focal adhesions. In turn, locally elevated Ca2+ ion levels activates and recruits S100A11 to the adhesion sites targeted for disassembly. 

So how did they work this out?

The force-dependent recruitment of S100A11 to stressed focal adhesions was confirmed using a micropipette pulling assay able to apply pulling forces onto individual focal adhesion sites. Even when myosin II-dependent intracellular contractility was inhibited, external pulling forces still recruited S100A11 to stretched focal adhesion sites, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ ion and Piezo1 function was still indispensable, indicating that myosin II-dependent contraction forces act upstream of Piezo1-mediated Ca2+ ion influx, in turn leading to S100A11 activation and focal adhesion recruitment.

Lastly, the authors show impaired focal adhesion translocation and disassembly rates in S100A11 knockout cells, confirming the important role of S100A11 in focal adhesion turnover. Together,&

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Researchers observe what ubiquitination hinges on 

Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Hiroki Konno and Holger Flechsig at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University.

The research described in this podcast was published in Nano Letters in December 2023

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https://nanolsi.kanazawa-u.ac.jp/en/

Researchers observe what ubiquitination hinges on

Researchers at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University report in Nano Letters how the flexibility of a protein hinge plays a crucial role in the transfer of proteins in key cell processes.

Ubiquitination – the addition of the protein ubiquitin – is a key stage in many cell processes, such as protein degradation, DNA repairs, and signal transduction. Using high-speed atomic force microscopy (AFM) and molecular modelling, researchers led by Hiroki Konno and Holger Flechsig at WPI-NanoLSI, Kanazawa University have identified how the mobility of a ubiquitination related enzyme hinge allows ubiquitination to take place.

So what was known already about ubiquitination?

Previous studies have identified a number of enzymes that facilitate ubiquitination, including an enzyme that activates ubiquitin (E1), an enzyme that conjugates it (E2), and an enzyme that catalyzes ubiquitin protein joining (that is, a ligase, E3) to a target protein. The HECT-type E3 ligase is characterized by a HECT domain that comprises an N lobe with the E2-binding site and a C lobe with a catalytic Cys residue, A flexible hinge connects the two lobes, leading to the hypothesis that ubiquitination is facilitated by the rearrangement of the protein around this hinge. Konno and their collaborators deployed their high-speed atomic force microscope to hunt for evidence that this was the case.

So what did they find out?

The researchers noted that when the HECT domain was crystallized with a type of E2 enzyme, it formed an L shape such that the distance between the catalytic Cys residue of the HECT domain and the catalytic Cys of the E2 enzyme was 41 Å – too far for the transfer of ubiquitin. However, in its catalytic conformation the HECT domain has a different shape where the distance between the two catalytic Cys residues is much closer – just 8 Å – so this is thought to be a “catalytic conformation”.

Analysis of high-speed-AFM images of a wild-type HECT domain of E6AP revealed two conformations – one of which looked spherical and the other oval. Using AFM simulations they attributed the oval shapes to the L conformation and spherical shapes are either the catalytic conformation or the so called inverted T conformation, which had been observed in the another type of HECT domain where the distance between the Cys residues is 16 Å. To overcome the spatio-temporal resolution limitations of imaging, the experiments were complemented by molecular modelling to visualize HECT domain conformational motions at the atomistic level. Simulation AFM was used to generate a corresponding pseudo AFM movie, which clearly showed the change from spherical to the oval shaped topography.

“Although experimental limitations do not allow us to resolve the intermediate conformations,” explain the researchers in their report of the work. “The performed modeling provides evidence that the transitions between spherical and oval HECT domain shapes observed under high-speed-AFM correspond to functional conformational motions under which the C-lobe rotates relative to the N-lobe, thereby allowing the change between catalytic and L-shape HECT conformations.”

Further experiments with mutant HECT domains with less flexibility in the hinge revealed no flipping between conformations – the mutant HECT domains were locked in the catalytic co

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Chromatin Accessibility: A new avenue for gene editing

Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, led by Yusuke Miyanari.

The research described in this podcast was published in Nature Genetics in February 2024

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https://nanolsi.kanazawa-u.ac.jp/en/

Chromatin Accessibility: A new avenue for gene editing

In a study recently published in Nature Genetics, researchers from Nano Life Science Institute (WPI-NanoLSI) at Kanazawa University explore chromatin accessibility, that is, endogenous access pathways to the genomic DNA, and its use as a tool for gene editing.

Our DNA is protected from unwanted external modifications by forming structures called nucleosomes that consist of threads of DNA wound around chunks of special proteins known as histones. This unique coiled shape prevents the access of undesirable molecules to a cell’s DNA. However, for vital genetic functions—such as DNA repair—the right set of proteins require access to these DNA fragments. This phenomenon known as ‘chromatin accessibility’ involves a privileged set of protein molecules, many of which are still unknown.

Now, researchers from Nano Life Science Institute (WPI-NanoLSI) at Kanazawa University, led by Yusuke Miyanari, have used advanced genetic screening methods to unravel chromatin accessibility and its pathways.

So how did they go about it?

For the investigation the team used a combination of two technologies—CRISPR screening and ATAC-see. While the former is a method to suppress the function of a desired set of genes, the latter is a means to identify which ones are essential for chromatin accessibility. Thus, using this method all genes playing a crucial role in chromatin accessibility could be pinned down.

With the help of these assays, novel pathways and individual players involved in chromatin accessibility were uncovered—some playing a positive role and some negative. Of these, one particular protein, TFDP1, showed a negative effect on chromatin accessibility. When it was suppressed, a significant increase in chromatin accessibility was observed, accompanied by nucleosome reduction. A deeper dive into the mechanism of TFDP1 revealed that it functions by regulating the genes responsible for production of certain histone proteins.

The team then focused their study towards exploring biotechnological applications of their findings. After suppressing TFDP1, two different approaches were tried. The first approach involved gene editing using the CRISPR/Cas9 tool. This revealed that deletion of TFDP1 made the gene editing process easier. Now, most chromatin accessibility occurs in nucleosome-depleted regions or NDRs. However, by suppressing TFDP1 chromatin accessibility occurred not only in NDRs but across other regions as well. Secondly, the depletion of TFDP1 aided the process of converting regular cells into stem cells, a massive step forward in cellular transformation.

This study identified new chromatin accessibility pathways and channels for exploring its potential even further. “Our study shows the significant potential to manipulate chromatin accessibility as a novel strategy to enhance DNA-templated biological applications, including genome editing and cellular reprogramming,” conclude the researchers.

Reference

Satoko Ishii, Taishi Kakizuka, Sung-Joon Park, Ayako Tagawa, Chiaki Sanbo, Hideyuki Tanabe, Yasuyuki Ohkawa, Mahito Nakanishi, Kenta Nakai, Yusuke Miyanari. Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility. Nature Genetics, Feb 15, 2024 . 

https://www.nature.com/articles/s41588-024-01658-1

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